Immunization with wild-type sequence (wt) p53 epitopes represents a novel therapeutic strategy for cancer patients. We have used autologous dendritic cells (DC) pulsed with wt p53(264-272) peptide to generate p53 peptide-specific T cells from peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), as well as healthy donors. T cells of 30 patients and 31 healthy individuals were evaluated by multicolor flow cytometry using peptide-HLA-A2.1 complexes. Patients with SCCHN had a significantly higher mean frequency of CD8+ T cells specific for wt p53(264-272) than normal donors (P = 0.0041). In patients whose tumors had normal p53 expression or had p53 gene mutations preventing presentation of this epitope, high frequencies of wt p53(264-272)-specific CD8+ memory T cells were found. In contrast, patients whose tumors accumulated p53 had low frequencies of wt p53(264-272)-specific naive CD8+ T cells that were unable to proliferate ex vivo in response to the epitope. This seemingly contradictory relationship between the high frequency of epitope-specific T cells and wt p53 expression in the tumor suggests that other factors may contribute to the observed anti-p53 responses. To increase the responsive rate to the wt p53(264-272) peptide, we sought to identify more immunogenic variants of this peptide. Two such variants induced T cells from PBMC of nonresponsive healthy and SCCHN donors that recognized the parental peptide either pulsed onto target cells or naturally presented by tumors. TCR Vb analysis confirms the heteroclitic nature of at least one of the variant peptides. The use of variant peptides of wt p53(264-272) represents a promising approach to overcoming the nonresponsiveness of certain cancer patients to this self epitope, thereby enhancing its potential use in tumor vaccines for appropriately selected cancer patients. Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8+ IFN-g and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-g in vitro recall responses were detected for all epitopes included in the construct. The relative immunogenicity of the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization were determined. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8+ IFN-g and HTL responses. We are continuing work on T cell biology using a xenoreactive mouse T cell clone, AHIII12.2, as a model system. During the past year, we have functionally characterized this T cell clone when responding to mouse and human pMHCs. We discovered that both pMHCs evoke similar T cell responses, and both recruit CD8 to the T cell synapse during T cell activation. We believe this suggests that high affinity TCR engagement can compensate for CD8 independence. Yet it is interesting that other alloreactive TCR recognize their allogeneic pMHC independently of CD8 and with high affinity. We believe this implies a general mechanism for T cell cross-reactivity, and intend to test this theory using several other cross-reactive TCR. Recently, we have crystallized the soluble TCR from this T cell clone with its xenoreactive pMHC. The crystal structure reveals a new binding mode with the TCR atop the pMHC, which is in sharp contrast to the 12 TCR:pMHC structures determined to date. As the xenoreactive TCR was not selected during T cell development on the human pMHC, the unusual orientation of this xenoreactive TCR:pMHC complex suggests an orientation requirement for TCR to survive positive selection during development. We plan to test this theory after creation of a xenoreactive TCR transgenic mouse.